This research is aimed at elucidating the collective molecular events of cellular signaling that are initiated by plasma membrane immunoreceptors and are highly orchestrated in space and time. Focus is primarily on the high affinity receptor for IgE, Fc5RI, on mast cells, which plays a central role in the allergic immune response and serves as a valuable model system for these studies. The goal of these studies is to understand structural interactions of proteins and lipids occurring within the plasma membrane environment that are altered by antigen crosslinking of IgE-Fc5RI and result in transmembrane triggering of the intracellular signaling cascade. The central hypothesis is that interactions between crosslinked IgE-Fc5RI, Lyn kinase, and other early signaling components are coordinated within the heterogeneous structure of the membrane including its interactions with the cytoskeleton. Proposed studies will investigate the roles of dynamic membrane structure and interactions in Fc5RI signaling, and, in particular, the participation of ordered membrane domains (commonly called rafts). Specific Aim 1 will continue to develop chemically modified substrates for patterning antigen on micro- and nanometer lengths scales, including use of thermosensitive polymers for rapidly triggered exposure of antigen. Spatially controlled, micron scale assemblies of co- clustered IgE-Fc5RI and cytoskeleton will be investigated as structures involved in initiation of internalization and phagocytosis. Specific Aim 2 will examine spatial regulation and dynamics of IgE-Fc5RI-mediated signaling assemblies on the nanoscale with scanning electron microscopy and super high-resolution fluorescence microscopy, and apertures for near-field optical microscopy will be nanofabricated as a novel approach for measuring motional and interactional dynamics of individual membrane components. Together with data analysis, a theoretical framework will be developed to describe how clustered receptors synchronize their activation state via the membrane. Specific Aim 3 will focus on participation of lipids in the membrane structural assemblies that spatially regulate IgE-Fc5RI mediated signaling using high resolution microscopies and electron spin resonance measurements of phase-like properties. Changes in proximity between Fc5RI and order- vs disorder- preferring lipids will be measured with fluorescence resonance energy transfer. Cholesterol distribution across the outer and inner leaflets of the plasma membrane will be assessed, and how perturbations of this distribution affect cell signaling events will be evaluated. These investigations integrate diverse physical, chemical and cell biological approaches to provide new insights into plasma membrane structure and dynamics and their roles in immunoreceptor signaling.